Refrigeration, particularly freezing, is a common and preferred means for storing biological materials. Frozen storage generally arrests or considerably slows the deterioration of the biological product.
Frozen or refrigerated foods are now a mainstay of the human diet in developed nations. Thus extensive research has and is being carried out by food scientists to ensure high quality products for the consumers. This is particularly true with regard to frozen vegetables and frozen deserts such as ice cream and yogurt.
Frozen deserts such as ice cream or yogurt are generally eaten in the frozen state. Thus, the texture of the frozen product as well as its flavor is important to consumers. Texture is to a large extent governed by the size of the ice crystals. Producers of these frozen deserts have gone to considerable effort and expense to ensure smooth textured products. However, during frozen storage the ice crystals can grow and thus roughen and spoil this texture. The growth of ice crystals during frozen storage is known as recrystallization. This problem is particularly common when the frozen storage conditions are less than ideal, such as during transportation or storage in modern frost-free home freezers. After a relatively short period of time at above-zero temperatures (i.e., above 0.degree. C.), or even at sustained freezing temperatures, frozen foods can become less desirable or even unsuitable for human consumption due to the ice recrystallization process.
Although manufacturers use a variety of techniques to reduce the damage associated with recrystallization success has been limited and significant problems remain. Thus there is a need for new techniques to reduce or prevent the recrystallization process and improve the characteristics of frozen foods. These techniques and compositions should be inexpensive and completely safe and suitable for human consumption.
It has been clearly demonstrated that antifreeze polypeptides (AFP) can effectively inhibit ice recrystallization at low (.mu.g/ml) concentrations in aqueous solutions and frozen food products (see, e.g., Knight et al., 1984, Nature 308:295-296; Knight et al., 1986, Cryobiology, 23:256-262; Knight et al., 1988, Cryobiology, 25:55-60; Warren et al., U.S. Pat. No. 5,118,792). Warren et al, supra, have suggested adding purified antifreeze polypeptides directly to food products prior to freezing to improve preservation characteristics during frozen storage.
At the present time antifreeze proteins are available for commercial use from two sources; the blood serum from a small number of fish species found in cold water, and recombinant DNA techniques such as those described by (but not restricted to) Warren et al., supra. Other sources of antifreeze proteins, such as transgenic plants and animals, are currently being explored. Regardless of the source, the antifreeze polypeptides must be isolated from the medium in which they are found or produced, and subject to extensive purification. These purification procedures are expensive to the point where the cost of the antifreeze polypeptide additions could exceed the value of the frozen product. Moreover, the purification protocol may introduce contaminants unsafe for consumption.
In the view of the inherent value to producers and consumers of inhibiting ice recrystallization in frozen fermented dairy products, and the fact that antifreeze polypeptides are very effective in this regard, it is desirable to develop methods to incorporate the antifreeze polypeptides into the food products in the most efficient and cost effective method possible.
An ideal method of incorporating antifreeze polypeptides into frozen fermented food products is to have the organism responsible for the fermentation process produce the antifreeze proteins while fermenting the food. A number of antifreeze polypeptides and their genes have been well characterized and sequenced (see, e.g. Anathanarayanan, 1989, Life Chem. Rep. 7:1-32 and Davies et al., 1990, FASEB Journal 4:2460-2467). Several of these genes have been expressed in bacteria, transgenic plants, fish and Drosophila (see, e.g., Fletcher et al., 1988, Can. J. Fish. Aquat. Sci. 45:352-357; Rancourt et al., 1987, Mol. Cell. Biol. 7:2188-2195; Kenward et al., 1993, Plant Mol.Biol. 23:377-385; Li et al., 1991, Protein Engineering 4:995-1012; and Sonnichsen et al., 1993, Science 259:1154-1157).
In view of the widespread popularity of frozen fermented dairy products such as yogurt it would be desirable to develop methods for producing such products more efficiently at lower cost and with better flavor and texture. The present invention provides methods for achieving this goal.